Tyrosine DNA phosphodiesterases (TDP) and related polypeptides nucleic acids vectors TDP producing host cells antibodies and methods of use

ABSTRACT

The present invention provides a nucleic acid molecule encoding a tyrosine-DNA phosphodiesterase (TDP), and a related vector, host cell, polypeptide, antibody, antisense nucleic acid molecule, and ribozyme. Also provided are a method of altering the level of TDP in a cell, tissue, organ or organism, as well as the resulting cell, tissue, organ or non-human organism, as well as a method of identifying a TDP-resistant compound, a method of assessing TDP1 activity in an animal, and a method of assessing the efficacy of a topoisomerase I inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase of PCT/US00/27400, which wasfiled on Oct. 5, 2000, and which claims priority to U.S. provisionalapplication Ser. No. 60/157,690, which was filed on Oct. 5, 1999.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to tyrosine-DNA phosphodiesterases andrelated polypeptides, nucleic acids, vectors, TDP-producing host cells,antibodies and methods of use in the identification of a TDP-resistantcompound, in the assessment of TDP1 activity in an animal, and in theassessment of efficacy of a topoisomerase I inhibitor.

BACKGROUND OF THE INVENTION

Topoisomerases are cellular enzymes that are crucial for replication andtranscription of the cellular genome. Topoisomerases cleave the DNAbackbone, thereby allowing topological change for replication andtranscription of the cellular genome to occur, after whichtopoisomerases reseal the DNA backbone (Wang, Ann. Rev. Biochem. 65: 635(1996)). Topoisomerases are efficient because DNA breakage isaccompanied by covalent bonding between the enzyme and the DNA to createan intermediate that is resolved during the resealing step. Thismechanism, while elegant, makes topoisomerases potentially dangerous. Ifthe resealing step fails, a normally transient break in DNA becomes along-lived disruption, one with a topoisomerase covalently joined to it.Unless a way is found to restore the continuity of the DNA the cell willdie.

In virtually all topoisomerases, the heart of the covalent complex is aphosphodiester bond between a specific tyrosine residue of the enzymeand one end of the break (the 3′ end for eukaryotic topoisomerase I andthe 5′ end for topoisomerases II and III). The high-energy nature ofthis bond normally ensures the resealing step.

Failure of resealing is dramatically increased by several drugs, such ascamptothecin, a promising anti-cancer agent that specifically targetseukaryotic topoisomerase I (Chen et al., Ann. Rev. Pharmacol. Toxicol.34: 191 (1994)). Protein-linked breaks also accumulate whentopoisomerases act on DNA containing structural lesions like thyminedimers, abasic sites and mismatched base pairs (Pommier et al., Biochim.Biophys. Acta 1400: 83 (1998)). To the extent that such lesions ariseduring the normal life of a cell, topoisomerase-associated damage may beunavoidable.

Repair of topoisomerase-DNA covalent complexes is of obvious value tothe cell but, until the present invention, very little was known aboutthe mechanisms involved in such repair. Hydrolysis of the bond joiningthe topoisomerase to DNA had been proposed as a way to effect release ofthe topoisomerase such that the cleaved DNA could undergo conventionalmodes of break repair (Friedberg et al., DNA Repair and Mutagenesis (ASMPress, Washington, D.C. (1995)); Kanaar et al., Trends Cell. Biol. 8:483 (1998)). Although no such hydrolysis has been reported for covalentcomplexes between DNA and topoisomerase II or III, such hydrolysis hasbeen described for covalent complexes between DNA and topoisomerase I(Yang et al., PNAS USA 93: 11534 (1996)).

The present invention seeks to provide the enzyme responsible forhydrolysis of the covalent complexes between DNA and topoisomerase I,specifically tyrosine-DNA phosphodiesterase, which acts on a tyrosinelinked through the side-chain oxygen to the 3′ phosphate of DNA. Thisand other objects and advantages, as well as additional inventivefeatures, will become apparent to one of ordinary skill in the art fromthe detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated and purified nucleic acidmolecule consisting essentially of a nucleotide sequence encoding amammalian, in particular a human, tyrosine-DNA phosphodiesterase (TDP1)and a continuous fragment thereof of at least about 36 nucleotides. Alsoprovided is an isolated or purified nucleic acid molecule encoding amodified mammalian TDP1, which comprises one or more insertions,deletions and/or substitutions, and a continuous fragment thereof of atleast about 36 nucleotides. The modified mammalian TDP1 does not differfunctionally from the corresponding unmodified mammalian TDP1.

An isolated and purified nucleic acid molecule consisting essentially ofa nucleotide sequence encoding a yeast, in particular a Saccharomycescerevisiae, TDP1 and a continuous fragment thereof comprising at leastabout 36 nucleotides are also provided by the present invention. In thisregard, an isolated or purified nucleic acid molecule encoding amodified yeast TDP1, which comprises one or more insertions, deletionsand/or substitutions, and a continuous fragment thereof of at leastabout 36 nucleotides, are also provided by the present invention. Themodified yeast TDP1 does not differ functionally from the correspondingunmodified yeast TDP1.

The present invention further provides a vector comprising anabove-described nucleic acid molecule and a vector comprising orencoding an antisense molecule of at least about 20 nucleotides thathybridizes to or a ribozyme that cleaves an RNA molecule encoding anabove-described TDP1, as well as the antisense molecule and theribozyme.

Also provided by the present invention is a host cell comprising anabove-described vector, a polypeptide produced by such a host cell, anda polyclonal or monoclonal antibody that binds to an above-describedTDP1.

Further provided by the present invention is a method of altering thelevel of TDP1 in a cell, a tissue, an organ or an organism. The methodcomprises contacting a cell, a tissue, an organ or an organism with avector comprising a (i) a nucleic acid molecule encoding a TDP1, (ii) anucleic acid molecule comprising or encoding an antisense molecule of atleast about 20 nucleotides to an RNA molecule transcribed from (i), or(iii) a nucleic acid molecule comprising or encoding a ribozyme to anRNA molecule transcribed from (i). The vector comprising (i) increasesor decreases the level of TDP1 in the cell, the tissue, the organ or theorganism, whereas the vector comprising (ii) or (iii) decreases thelevel of TDP1 in the cell, the tissue, the organ or the organism. Inthis regard, the present invention also provides a cell, a tissue, anorgan or a nonhuman organism in which the level of TDP1 has been alteredin accordance with such a method.

Another method provided by the present invention is a method ofidentifying a compound that stabilizes a covalent bond complex thatforms between DNA and topoisomerase I, wherein the covalent bond cannotbe cleaved by or is resistant to cleavage by a TDP1. The methodcomprises (a) contacting a compound, which stabilizes a covalent bondcomplex that forms between DNA and topoisomerase I such that thecovalent bond cannot be cleaved by or is resistant to cleavage by aTDP1, with DNA and topoisomerase I under conditions suitable for acovalent bond complex to form between the DNA and the topoisomerase Iand for the compound to stabilize the covalent bond complex, (b)contacting the covalent bond complex with a TDP1 under conditionssuitable for the cleavage of the covalent bond between the DNA andtopoisomerase I by TDP1, and (c) detecting cleavage of the covalent bondby the TDP1. The amount of cleavage detected is indicative of whether ornot the compound stabilizes a covalent bond complex that forms betweenDNA and topoisomerase I such that the covalent bond cannot be cleaved oris resistant to cleavage by the TDP1.

Yet another method provided by the present invention is a method ofassessing TDP1 activity in an animal. The method comprises (a) obtaininga sample of a cellular extract from an animal, wherein the cellularextract comprises TDP1, and (b) measuring the level of TDP1 activity inthe sample.

Still yet another method provided by the present invention is a methodof assessing the efficacy of a topoisomerase I inhibitor. The methodcomprises (a) obtaining a sample of DNA to which is covalently boundtopoisomerase I after contact with a topoisomerase I inhibitor, (b)contacting the sample with a TDP1 under conditions suitable for cleavageof the covalent bond between the DNA and the topoisomerase I by TDP1,and (c) measuring the amount of topoisomerase I that is cleaved from theDNA. The amount of topoisomerase I that is cleaved from the DNA isindicative of the efficacy of the topoisomerase I inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the cDNA sequence encoding a human TDP1 (SEQ ID NO: 1).

FIG. 2 is the deduced amino acid sequence (SEQ ID NO: 13) of the cDNA ofFIG. 1. The start codon (M) is circled.

FIG. 3 is the genomic DNA sequence encoding a yeast TDP1 (SEQ ID NO: 3).

FIG. 4 is the deduced amino acid sequence (SEQ ID NO: 4) of the genomicDNA of FIG. 3.

FIG. 5 is an alignment of TDP1 homologs from various organisms,including human (SEQ ID NO: 5), mouse (SEQ ID NO: 6), Drosophilamelanogaster (SEQ ID NO: 7), Caenorhabditis elegans (SEQ ID NO: 8),Schitosaccharomyces pombe (SEQ ID NO: 9), and Saccharomyces cerevisiae(SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides isolated or purifiednucleic acid molecules. By “isolated” is meant the removal of a nucleicacid from its natural environment. By “purified” is meant that a givennucleic acid, whether one that has been removed from nature (includinggenomic DNA and mRNA) or synthesized (including cDNA) and/or amplifiedunder laboratory conditions, has been increased in purity, wherein“purity” is a relative term, not “absolute purity.” “Nucleic acidmolecules” is intended to encompass a polymer of DNA or RNA, i.e., apolynucleotide, which can be single-stranded or double-stranded andwhich can contain non-natural or altered nucleotides.

One isolated or purified nucleic acid molecule consists essentially of anucleotide sequence encoding a mammalian TDP1 or a continuous fragmentthereof of at least about 36 nucleotides. Preferably, the mammalian TDP1is a human TDP1. Also, preferably, the mammalian TDP1 is (i) DNA andconsists essentially of SEQ ID NO: 1 or a sequence that encodes SEQ IDNO: 2, (ii) RNA and consists essentially of a sequence encoded by SEQ IDNO: 1 or a sequence that encodes SEQ ID NO: 2, or (iii) a nucleic acidmolecule consisting essentially of a nucleotide sequence that encodes amammalian TDP1 or a continuous fragment of at least about 12 amino acidsthereof and that hybridizes to either one of the foregoing under lowstringency conditions.

Also provided is an isolated or purified nucleic acid molecule encodinga modified mammalian TDP1, which comprises one or more insertions,deletions and/or substitutions, wherein the modified mammalian TDP1encoded by the isolated or purified nucleic acid molecule does notdiffer functionally from the corresponding unmodified mammalian TDP1, ora continuous fragment thereof of at least about 36 nucleotides.Desirably, the modified mammalian TDP1 does not differ functionally fromthe corresponding unmodified mammalian TDP1, such as that comprising SEQID NO: 2. Preferably, the modified mammalian TDP1 cleaves a covalentbond complex between DNA and topoisomerase I at least about 90% as wellas the corresponding unmodified mammalian TDP1, such as that comprisingSEQ ID NO: 2, as determined by in vitro assay using labeledtopoisomerase I or an oligonucleotide comprising a 3′ phosphotyrosine(see, e.g., Yang et al. (1996), supra). Use of the word “labeled” hereinis intended to mean any means of detection, such as a radioactiveisotope.

Another isolated or purified nucleic acid molecule consists essentiallyof a nucleotide sequence encoding a yeast TDP1 or a continuous fragmentthereof comprising at least about 36 nucleotides. Preferably, the yeastTDP1 is a Saccharomyces cerevisiae TDP1. Also, preferably, the yeastTDP1 is (i) DNA and consists essentially of SEQ ID NO: 3 or a sequencethat encodes SEQ ID NO: 4, (ii) RNA and consists essentially of asequence encoded by SEQ ID NO: 3 or a sequence that encodes SEQ ID NO:4, or (iii) a nucleic acid molecule consisting essentially of anucleotide sequence that encodes a yeast TDP1 or a continuous fragmentof at least about 12 amino acids thereof and that hybridizes to eitherone of the foregoing under low stringency conditions. Yeast TDP1 willact on a tyrosine linked through the side-chain oxygen to the 3′phosphate of single-stranded and double-stranded DNA.

Also provided is an isolated or purified nucleic acid molecule encodinga modified yeast TDP1, which comprises one or more insertions, deletionsand/or substitutions, wherein the modified yeast TDP1 encoded by theisolated or purified nucleic acid molecule does not differ functionallyfrom the corresponding unmodified yeast TDP1, or a continuous fragmentthereof of at least about 36 nucleotides. Desirably, the modified yeastTDP1 does not differ functionally from the corresponding unmodifiedyeast TDP1, such as that comprising SEQ ID NO: 4. Preferably, themodified yeast TDP1 cleaves a covalent bond complex between DNA andtopoisomerase I at least about 90% as well as the correspondingunmodified yeast TDP1, such as that comprising SEQ ID NO: 4, asdetermined by in vitro assay using labeled topoisomerase I or anoligonucleotide comprising a 3′ phosphotyrosine.

With respect to the above, one of ordinary skill in the art knows how togenerate insertions, deletions and/or substitutions in a given nucleicacid molecule. Also with respect to the above, “does not differfunctionally from” is intended to mean that the modified enzyme hasenzymatic activity characteristic of the unmodified enzyme. In otherwords, it acts upon the same substrate and generates the same product.The modified enzyme, however, can be more or less active than theunmodified enzyme as described in accordance with the present invention.

Nucleic acid molecules encoding TDP1 can be isolated from numerouseukaryotic sources. In this regard, TDP1 is highly conserved amongeukaryotes. With respect to the above isolated or purified nucleic acidmolecules, it is preferred that the one or more substitution(s) do(es)not result in a change in an amino acid of the enzyme. Alternatively,and also preferred, is that the one or more subsitution(s) result(s) inthe substitution of an amino acid of the encoded yeast TDP1 with anotheramino acid of approximately equivalent size, shape and charge.

Also with respect to the above isolated or purified nucleic acidmolecules, a “continuous fragment of at least about 36 nucleotides ofthe isolated or purified nucleic acid molecule,” preferably encodes apolypeptide that can carry out the same function as the correspondingcomplete polypeptide or protein. For example, a fragment of an isolatedor purified nucleic acid molecule encoding a mammalian TDP1 can be acontinuous fragment of the TDP1-encoding nucleic acid molecule thatencodes a polypeptide that can cleave a covalent bond complex betweenDNA and topoisomerase I, but not necessarily as well as thecorresponding complete polypeptide or protein.

The above isolated or purified nucleic acid molecules also can becharacterized in terms of “percentage of sequence identity.” In thisregard, a given nucleic acid molecule as described above can be comparedto a nucleic acid molecule encoding a corresponding gene (i.e., thereference sequence) by optimally aligning the nucleic acid sequencesover a comparison window, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence, which does notcomprise additions or deletions, for optimal alignment of the twosequences. The percentage of sequence identity is calculated bydetermining the number of positions at which the identical nucleic acidbase occurs in both sequences, i.e., the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Optimal alignment ofsequences for comparison can be conducted by computerizedimplementations of known algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis., or BlastN and BlastXavailable from the National Center for Biotechnology Information,Bethesda, Md.), or by inspection. Sequences are typically compared usingBESTFIT or BlastN with default parameters.

“Substantial sequence identity” means that at least about 75%,preferably at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% of the sequence of a given nucleicacid molecule is identical to a given reference sequence or that atleast about 40%, preferably at least about 60%, more preferably at leastabout 90%, and most preferably at least about 95% of the amino acids ofwhich a given polypeptide is comprised are identical to or representconservative substitutions of the amino acids of a given referencesequence.

Another indication that polynucleotide sequences arc substantiallyidentical is if two molecules selectively hybridize to each other understringent conditions. The phrase “selectively hybridizing to” refers tothe selective binding of a single-stranded nucleic acid probe to asingle-stranded target DNA or RNA sequence of complementary sequencewhen the target sequence is present in a preparation of heterogeneousDNA and/or RNA. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 20° C. lower than the thermal melting point(Tm) for the specific sequence at a defined ionic strength and pH. TheTm is the temperature (under defined ionic strength and pH) at which 50%of the target sequence hybridizes to a perfectly matched probe. “Lowstringency conditions,” as that term is used herein, means thoseconditions that allow for as much as about 80% mismatch.

The above-described nucleic acid molecules can be used, in whole or inpart (i.e., as fragments), to identify and isolate corresponding genesfrom other eukaryotes for use in the context of the present inventivemethod using conventional means as known in the art. For example, suchmolecules or fragments thereof can be used in chromosome walking,genomic subtraction, which requires the availability of strains havingdeletions of the target gene (Strauss and Ausubel, PNAS USA 87:1889-1893 (1990); and Sun et al., Plant Cell 4: 119-128 (1992)),transposon (Chuck et al., Plant Cell 5: 371-378 (1993); Dean et al.,Plant J. 2: 69-81 (1992); Grevelding et al., PNAS USA 899: 6085-6089(1992); Swinburne et al., Plant Cell 4: 583-595 (1992); Fedoroff andSmith, Plant J. 3: 273-289 (1993); and Tsay et al., Science 260: 342-344(1993)) and T-DNA tagging (Feldmann, Plant J. 1: 71-82 (1991); Feldmannet al., Science 243: 1351-1354 (1989); Herman et al., Plant Cell 11:1051-1055 (1989); Konz et al., EMBO J. 9: 1337-1346 (1989); and Kieberet al., Cell 72: 427-441 (1993)), and heterologous probe selectiontechniques in accordance with methods well-known in the art. AlthoughT-DNA tagging, chromosome walking or heterologous probe selection canidentify a DNA fragment that putatively contains the gene of interest,the DNA fragment must be confirmed by genetic complementation or someother means.

In another embodiment, the present invention also provides a vectorcomprising a nucleic acid molecule as described above. A nucleic acidmolecule as described above can be cloned into any suitable vector andcan be used to transform or transfect any suitable host cell, whether asingle cell or a collection of cells, such as in the context of atissue, an organ or an organism. The selection of vectors and methods toconstruct them are commonly known to persons of ordinary skill in theart and are described in general technical references (see, in general,“Recombinant DNA Part D,” Methods in Enzymology, Vol. 153, Wu andGrossman, eds., Academic Press (1987)). Desirably, the vector comprisesregulatory sequences, such as transcription and translation initiationand termination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid sequence as described above or a portionthereof ligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from ColEl, 2mμ plasmid, λ, SV40, bovine papilloma virus, and the like. Yeastcentromeric plasmid (YCp50) constructs can be used to express TDP1 inyeast.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts, if so desired. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like.

Suitable vectors include those designed for propagation and expansion orfor expression or both. A preferred cloning vector is selected from thegroup consisting of the pUC series, the pBluescript series (Stratagene,LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEXseries (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clonetech, Palo Alto, Calif.). Bacteriophage vectors, such as λ GT10,λGT11, λZapII (Stratagene), λ EMBL4, and λ NM1149, also can be used.Examples of plant expression vectors include pBI101, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clonetech, Palo Alto, Calif.). Examples of animalexpression vectors include pEUK-C1, pMAM and pMAMneo (Clonetech, PaloAlto, Calif.). Other examples of suitable vectors include the yeastcentromeric plasmid (YCp50), 2u and integrative vectors, such as the pRSseries (Bachmann et al., Yeast 14: 115 (1998)) or PYES2 (Invitrogen).

An expression vector can comprise a native or nonnative promoteroperably linked to a nucleic acid molecule encoding a TDP1 as describedabove. The selection of promoters, e.g., strong, weak, inducible,repressible, cell-specific, tissue-specific, organ-specific anddevelopmental-specific, is within the skill in the art. Similarly, thecombining of a nucleic acid molecule as described above with a promoteris also within the skill in the art.

The present invention not only provides a vector comprising a nucleicacid molecule as described above-but also provides a vector comprisingor encoding an antisense molecule that hybridizes to or a ribozyme thatcleaves an RNA molecule encoding a TDP1 as described above. The presentinvention also provides the antisense molecules and ribozymes,themselves.

Antisense nucleic acids can be generated in accordance with methodsknown in the art. The nucleic acid molecule introduced in antisenseinhibition generally is substantially identical to at least a portion,preferably at least about 20 continuous nucleotides, of the nucleic acidto be inhibited, but need not be identical. The complex can, thus, bedesigned such that the inhibitory effect applies to other proteinswithin a family of genes exhibiting homology or substantial homology tothe nucleic acid. The introduced sequence also need not be full-lengthrelative to either the primary transcription product or fully processedmRNA. Generally, higher homology can be used to compensate for the useof a shorter sequence. Furthermore, the introduced sequence need nothave the same intron or exon pattern, and homology of non-codingsegments will be equally effective.

The inclusion of ribozyme sequences within antisense RNAs confersRNA-cleaving activity upon them, thereby increasing the activity of theconstructs. The design and use of target RNA-specific ribozymes isdescribed in Haseloff et al., Nature 334: 585-591 (1988). Preferably,the ribozyme comprises at least about 20 continuous nucleotidescomplementary to the target sequence on each side of the active site ofthe ribozyme.

In view of the above, the present invention provides a host cellcomprising a vector as vector as described above. In addition, thepresent invention provides a polypeptide produced by a host cellcomprising a vector as described above.

Suitable hosts of cells include mammalian, such as human, and insectcells, yeast (e.g., S288c strain (wild-type) S. cerevisiae), andbacteria (e.g., E. coli strain BL21 (DE3)).

Also provided by the present invention is a purified and isolatedmammalian, in particular human, TDP1. A mammalian, in particular ahuman, TDP1 can be purified and isolated using methods known to those ofordinary skill in the art. For example, a human, TDP1 can be isolated orpurified by forming a human TDP1 fusion protein containing apolyhistidine tail and purifying via nickel-chelation chromatography.

In addition to the above, the present invention provides polyclonal andmonoclonal antibodies to TDP1. Preferably, the antibody binds to amammalian TDP1 but does not bind to a nonammalian TDP1 or the antibodybinds to a yeast TDP1 but does not bind to a non-yeast TDP1. Methods ofpolyclonal and monoclonal antibody production are known in the art. See,for example, Harlow and Lane, in Antibodies. A Laboratory Manual. ColdSpring Harbor Laboratory, Cold Spring Harbor, 1988, pp. 1-725). Forexample, a yeast TDP fusion protein containing a polyhistidine tail wasexpressed in a bacterial expression vector and purified vianickel-chelation chromatography. Pure protein was isolated frombacterial cultures and injected into rabbits using standard methods togenerate polyclonal antibodies. Antibodies were harvested ten weekslater and were shown to interact effectively with and to precipitateyeast TDP1.

In another embodiment, the present invention provides a method ofaltering the level of TDP1 in a cell, tissue, organ or organism. By“altering” is meant that the TDP1 level in a given cell, tissue, organor organism is different as a result of the practice of the presentinventive method as compared to a like cell, tissue, organ or organismin which the level of TDP1 has not been altered as a result of thepractice of the present inventive method.

The method comprises contacting the cell, the tissue, the organ or theorganism with a vector comprising a nucleic acid molecule selected fromthe group consisting of (i) a nucleic acid molecule encoding a TDP1,(ii) a nucleic acid molecule comprising or encoding an antisensesequence of at least about 20 nucleotides to an RNA molecule transcribedfrom (i), and (iii) a nucleic acid molecule comprising or encoding aribozyme to an RNA molecule transcribed from (i). The vector comprisinga nucleic acid molecule of (i) increases or decreases the level of TDP1in the cell, the tissue, the organ or the organism, whereas the vectorcomprising a nucleic acid molecule of (ii) or (iii) decreases the levelof TDP1 in the cell, the tissue, the organ or the organism. Accordingly,the present invention further provides a cell, a tissue, an organ or anonhuman organism in which the level of TDP1 has been altered inaccordance with the method.

By “contacting” is meant bringing the cell, tissue, organ or organisminto sufficiently close proximity with the vector such that the vectoris taken up by the cell or by cells in the tissue, organ or organism,wherein it can be expressed. The method is not dependent on anyparticular means of contact and is not to be so construed. Means ofcontact are well-known to those skilled in the art, and also areexemplified herein.

Accordingly, contact can be effected, for instance, either in vitro(e.g., in an ex vivo type method of gene therapy) or in vivo, whichincludes the use of electroporation, transformation, transduction,conjugation or triparental mating, transfection, infection, membranefusion with cationic lipids, high-velocity bombardment with DNA-coatedmicroprojectiles, incubation with calcium phosphate-DNA precipitate,direct microinjection into single cells, and the like. Other methodsalso are available and are known to those skilled in the art.

Preferably, however, the vectors (including antisense molecules andribozymes) are introduced by means of cationic lipids, e.g., liposomes.Such liposomes are commercially available (e.g., Lipofectin®,Lipofectamine™, and the like, supplied by Life Technologies, Gibco BRL,Gaithersburg, Md.). Moreover, liposomes having increased transfercapacity and/or reduced toxicity in vivo (e.g., as reviewed in PCTpatent application no. WO 95/21259) can be employed in the presentinvention. For liposome administration, the recommendations identifiedin the PCT patent application No. WO 93/23569 can be followed.Generally, with such administration the formulation is taken up by themajority of lymphocytes within 8 hr at 37° C., with more than 50% of theinjected dose being detected in the spleen an hour after intravenousadministration. Similarly, other delivery vehicles include hydrogels andcontrolled-release polymers.

The form of the vector introduced into a host cell can vary, dependingin part on whether the vector is being introduced in vitro or in vivo.For instance, the nucleic acid can be closed circular, nicked, orlinearized, depending on whether the vector is to be maintainedextragenomically (i.e., as an autonomously replicating vector),integrated as a provirus or prophage, transiently transfected,transiently infected as with use of a replication-deficient orconditionally replicating virus, or stably introduced into the hostgenome through double or single crossover recombination events.

Preferably, the nucleic acid molecule used in the above-described methodis one of those described above. In this regard, nucleic acid moleculesthat correspond to the above-described nucleic acid molecules but whichhave been isolated from other eukaryotic sources, in particular othermammalian or yeast sources, can be used in the context of the presentinventive method to increase the level of TDP1 in a cell, tissue, organor organism, provided that a cDNA sequence is used in those instanceswhere the genomic sequence contains introns that may not be properlyprocessed in a given cell, tissue, organ or organism. In addition, itmay be necessary to alter the cDNA sequence so that it contains codonsequences that are preferred in a given species. However, to the extentthat antisense or ribozyme sequences arc employed in the presentinventive method, it would be advantageous to use a nucleic acidmolecule isolated from a eukaryotic source that is of the same origin asthe cell, the tissue, the organ or the organism in which the level ofTDP1 is to be altered.

If it is desired to increase the expression of TDP1, it is preferred todo so by introducing a gene encoding TDP1. Preferably, a vectorcomprising a nucleotide sequence encoding TDP1 operably linked to apromoter that is functional in the cell, the tissue, the organ or theorganism with which it is brought into contact is used. It is preferredthat either multiple extra copies of the gene are introduced into thecell, the tissue, the organ or the organism or that a vector comprisinga strong promoter is introduced into the cell, the tissue, the organ orthe organism such that the coding sequence is expressed at a higherrate, thereby generating more mRNA, which, in turn, is translated intomore of the encoded enzyme.

In this regard, if expression is desired in a given cell, tissue ororgan, a cell-, tissue- or organ specific promoter can be used in thevector. Developmentally specific promoters and regulatable, i.e.,inducible or repressible, e.g., metallothionein promoter andradiation-responsive promoter, also can be used. Examples of suchpromoters, as well as enhancer elements and suppressor elements, areknown in the art. Promoters can be found, for example, in eukaryoticpromoter databases (see, e.g., the Eukaryotic Promoter Database of theSwiss Institute for Experimental Cancer Research (ISREC) (Epalinges,Switzerland) (available online through the Kyoto UniversityBioinformatics Center (GenomeNet) (Kyoto, Japan)) and other suchdatabases.

In addition, a nucleic acid can be directly or indirectly linked to atargeting moiety. A “targeting moiety,” such as that term is used hereinis any molecule that can be linked with an above-described nucleic aciddirectly or indirectly, such as through a suitable delivery vehicle,such that the targeting moiety preferentially binds to a target cell ascompared to a non-target cell. The targeting moiety can bind to a targetcell through a receptor, a substrate, an antigenic determinant oranother binding site on the target cell. Examples of a targeting moietyinclude an antibody (i.e., a polyclonal or a monoclonal antibody), animmunologically reactive fragment of an antibody, an engineeredimmunoprotein and the like, a protein (target is receptor, as substrate,or regulatory site on DNA or RNA), a polypeptide (target is receptor), apeptide (target is receptor), a nucleic acid, which is DNA or RNA (i.e.,single-stranded or double-stranded, synthetic or isolated and purifiedfrom nature; target is complementary nucleic acid), a steroid (target issteroid receptor), and the like. In general, there are a number ofdatabases for targeting moieties (see, e.g., the Kyoto Encyclopedia ofGenes and Genomes (available online through the Kyoto UniversityBioinformatics Center (GenomeNet) (Kyoto, Japan)); Kanehisa, TrendsGenet., 13, 375-376 (1997); and Kanehisa, et al., Nucleic Acids Res.,28, 27-30 (2000)).

Analogs of targeting moieties that retain the ability to bind to adefined target also can be used. In addition, synthetic targetingmoieties can be designed, such as to fit a particular epitope.Alternatively, the therapeutic nucleic acid can be encapsulated in aliposome comprising on its surface the targeting moiety.

The targeting moiety can include a linking group that can be used tojoin a targeting moiety to, in the context of the present invention, anabove-described nucleic acid. It will be evident to one skilled in theart that a variety of linking groups, including bifunctional reagents,can be used. The targeting moiety can be linked to the nucleic acid bycovalent or non-covalent bonding. If bonding is non-covalent, theconjugation can be through hydrogen bonding, ionic bonding, hydrophobicor van der Waals interactions, or any other appropriate type of binding.

If it is desired to decrease the expression of TDP1, it is preferred todo so by introducing either a nucleic acid molecule comprising (i.e., inthe case of an RNA vector) or encoding (i.e., in the case of a DNAvector) an antisense nucleic acid molecule to an RNA moleculetranscribed from an aforementioned gene or a nucleic acid moleculecomprising a ribozyme to an RNA molecule transcribed from such a gene.In antisense technology, a nucleic acid segment from the desired genecan be cloned and operably linked to the promoter sequence such that theanti-sense strand of RNA is transcribed. Another alternative method todecrease TDP1 is to use a compound that inhibits the transcription,translation or activity of TDP1.

In addition to the above, gene replacement technology can be used toincrease or decrease the expression of TDP1. Gene replacement technologyis based on homologous recombination. The nucleic acid of TDP1 can bemanipulated by mutagenesis (e.g., insertions, deletions, duplications orreplacements) to either increase or decrease its function. The alteredsequence can be introduced into the genome to replace the existing,e.g., wild-type, gene via homologous recombination.

The activity of TDP1 can be measured using labeled substrates in vitro.TDP1 activity can be assay quickly and conveniently in vitro by mixing aradioactively 5′-labeled 18-mer oligonucleotide with a 3′phosphotyrosylgroup and TDP1. The material is then run on a polyacrylamide sequencinggel and exposed to autoradiographic film. Product bands then can bequantitated (see Yang et al. (1996), supra). TDP1 will also act onthymidine 3′-nitrophenyl phosphate in vitro.

In addition to being useful in the study of TDP1, the above-describedmethod is useful in the context of prophylactic and therapeutictreatment, such as chemotherapy, in particular where thechemotherapeutic agent is an inhibitor of topoisomerase I and causes theformation of DNA-topoisomerase I complexes that cannot be cleaved or areresistant to cleavage by TDP1, such as campothecin, topotecan andirinotecan (CPT-11) and analogs thereof (see, e.g., Slichenmyer et al.,J. Natl. Cancer Inst. 85(4): 271-291 (1993); Slichenmyer et al., CancerChemother. Pharmacol. 34(Suppl.): S53-57 (1994); Burris & Fields,Hemtol. Onol. Clin. North Am. 8(2): 333-355 (1994); Hawkins, Oncology6(12): 17-23 (1992); Emerson et al., Cancer Res. 55(3): 603-609 (1995);Sugimori et al., J. Med. Chem. 37(19): 3033-3039 (1994); Wall et al., J.Med. Chem. 36(18): 2689-2700 (1993); Kingsbury et al., J. Med. Chem.34(1): 98-107 (1991); Wani et al., J. Med. Chem. 30(10): 1774-1779(1987); Wani et al., J. Med. Chem. 23: 554-560 (1980); and Wani et al.(1986)). In this regard, a patient with low levels of TDP1 might beoverly sensitive to compounds that inhibit topoisomerase I and mightbenefit from treatment with lower doses of topoisomerase I inhibitors orincreased expression of TDP1. In contrast, a patient with high levels ofTDP1 might be resistant to compounds that inhibit topoisomerase I andmight benefit from treatment with higher doses of topoisomerase Iinhibitors or reduced expression of TDP1.

In the context of chemotherapy and other methods of treatment, atopoisomerase I inhibitor and an above-described nucleic acid moleculecan be administered simultaneously or sequentially in either order, bythe same route of administration or by different routes ofadministration. In this regard, the topoisomerase I inhibitor and theabove-described nucleic acid molecule can be present in a singlebiologically or pharmaceutically acceptable composition or in separatebiologically or pharmaceutically acceptable compositions.Pharmaceutically acceptable compositions comprise pharmaceuticallyacceptable carriers and diluents as appropriate, for example, for humanor veterinary applications, as are known in the art.

Thus, a composition for use in the method of the present invention cancomprise an above-described nucleic acid molecule, e.g., vector,preferably in combination with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well-known to those skilled inthe art, as are suitable methods of administration. The choice ofcarrier will be determined, in part, by the particular nucleic acidmolecule, as well as by the particular method used to administer thecomposition. One skilled in the art will also appreciate that variousroutes of administering a composition are available and, although morethan one route can be used for administration, a particular route canprovide a more immediate and more effective reaction than another route.Accordingly, there are a wide variety of suitable formulations of thecomposition of the present invention.

A composition comprised of a nucleic acid molecule of the presentinvention, alone or in combination with another active agent, such as achemotherapeutic agent that inhibits topoisomerase I by causingformation of complexes between DNA and topoisomerase I that cannot-becleaved or are resistant to cleavage by TDP1, can be made into aformulation suitable for parenteral administration, preferablyintraperitoneal administration. Such a formulation can include aqueousand nonaqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit dose or multidose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneously injectable solutions and suspensions canbe prepared from sterile powders, granules, and tablets, as describedherein.

A formulation suitable for oral administration can consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid or granules; solutions or suspensions in an aqueousliquid; and oil-in-water emulsions or water-in-oil emulsions. Tabletforms can include one or more of lactose, mannitol, corn starch, potatostarch, microcrystalline cellulose, acacia, gelatin, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid,and other excipients, colorants, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, and pharmacologicallycompatible carriers.

An aerosol formulation suitable for administration via inhalation alsocan be made. The aerosol formulation can be placed into a pressurizedacceptable propellant, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Similarly, a formulation suitable for oral administration can includelozenge forms that can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier; as well as creams, emulsions, gels, and thelike containing, in addition to the active ingredient, such carriers asare known in the art.

A formulation suitable for topical application can be in the form ofcreams, ointments, or lotions.

A formulation for rectal administration can be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate. A formulation suitable for vaginal administration canbe presented as a pessary, tampon, cream, gel, paste, foam, or sprayformula containing, in addition to the active ingredient, such carriersas are known in the art to be appropriate.

The dose administered to a mammal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the infected individual over a reasonable time frame. Thedose will be determined by the potency of the particular vector employedfor treatment, the severity of the disease state, as well as the bodyweight and age of the infected individual. The size of the dose alsowill be determined by the existence of any adverse side effects that canaccompany the use of the particular vector employed. It is alwaysdesirable, whenever possible, to keep adverse side effects to a minimum.

The dosage can be in unit dosage form, such as a tablet or capsule. Theterm “unit dosage form” as used herein refers to physically discreteunits suitable as unitary dosages for human and animal subjects, eachunit containing a predetermined quantity of a vector, alone or incombination with other active agents, calculated in an amount sufficientto produce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier, or vehicle. The specifications for the unitdosage forms of the present invention depend on the particular compoundor compounds employed and the effect to be achieved, as well as thepharmacodynamics associated with each compound in the host. The doseadministered should be an “effective amount” or an amount necessary toachieve an “effective level” in the individual patient. Since the“effective level” is used as the preferred endpoint for dosing, theactual dose and schedule can vary, depending on interindividualdifferences in pharmacokinetics, drug distribution, and metabolism.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectivelevel” in the individual patient. One skilled in the art also canreadily determine and use an appropriate indicator of the “effectivelevel” of the compounds of the present invention by a direct (e.g.,analytical chemical analysis) or indirect analysis of appropriatepatient samples (e.g., blood and/or tissues).

Generally, an amount of vector sufficient to achieve a tissueconcentration of the administered ribozyme (or vector) of from about 50to about 300 mg/kg of body weight per day is preferred, especially offrom about 100 to about 200 mg/kg of body weight per day. In certainapplications, e.g., topical, multiple daily doses are preferred.Moreover, the number of doses will vary depending on the means ofdelivery and the particular vector administered. In the treatment ofsome individuals, it can be desirable to utilize a “mega-dosing”regimen.

In yet another embodiment, the present invention provides a method ofidentifying a compound that stabilizes a covalent bond complex thatforms between DNA and topoisomerase I such that the covalent bond cannotbe cleaved by or is resistant to cleavage by a TDP1. The methodcomprises (a) contacting a compound, which stabilizes a covalent bondcomplex that forms between DNA and topoisomerase I such that theconvalent bond cannot be cleaved by or is resistant to cleavage by aTDP1, with DNA and topoisomerase I under conditions suitable for acovalent bond complex to form between the DNA and the topoisomerase Iand for the compound to stabilize the covalent bond complex, (b)contacting the covalent bond complex with a TDP1 under conditionssuitable for cleavage of the covalent bond between the DNA andtopoisomerase I by TDP1, and (c) detecting cleavage of the covalent bondby the TDP1. The amount of cleavage detected is indicative of whether ornot the compound stabilizes a covalent bond complex that forms betweenDNA and topoisomerase I such that the covalent bond cannot be cleaved oris resistant to cleavage by the TDP1. For example, the more cleavagedetected, the less the compound stabilizes a covalent bond complex thatforms between DNA and topoisomerase I, and the less cleavage detected,the more the compound stabilizes a covalent bond complex that formsbetween DNA and topoisomerase I. Preferably, the compound is an analogof camptothecin, topotecan, or irinotecan (CPT- 11).

In still yet another embodiment, the present invention provides a methodof assessing TDP1 activity in an animal. The method comprises (a)obtaining a sample of a cellular extract from an animal, wherein thecellular extract comprises TDP1, and (b) measuring the level of TDP1activity in the sample. Assessing the level of TDP1 activity in apatient may be useful in predicting the patient's sensitivity to atopoisomerase I inhibitor, such as camptothecin, topotecan, irinotecan(CPT-11), an analog of any of the foregoing, and the like. For example,the more TDP1 activity a patient has, the less sensitive the patientwill be to a topoisomerase I inhibitor, and the less TDP1 activity apatient has, the more sensitive the patient will be to a topoisomerase Iinhibitor.

A still further embodiment of the present invention is a method ofassessing the efficacy of a topoisomerase I inhibitor. The methodcomprises (a) obtaining a sample of DNA to which is covalently boundtopoisomerase I after contact with a topoisomerase I inhibitor, (b)contacting the sample with a TDP1 under conditions suitable for cleavageof the covalent bond between the DNA and the topoisomerase I by TDP1,and (c) measuring the amount of topoisomerase I that is cleaved from theDNA. The amount of topoisomerase I that is cleaved from the DNA isindicative of the efficacy of the topoisomerase I inhibitor. Preferably,the sample of DNA is obtained from a patient undergoing treatment withthe topoisomerase I inhibitor and the patient's dosage or frequency ofadministration of topoisomerase I is adjusted down or up based on thehigh or low efficacy, respectively, of the topoisomerase I inhibitor.Also, preferably, the sample is obtained from peripheral blood cells ofthe patient. This method can be adapted for screening potentialenvironmental mutagens for DNA-topoisomerase I complexes that arenoncleavable by or resistant to cleavage by TDP1.

With respect to the above three methods, methods of contacting acompound with DNA and an enzyme, the determination of conditionssuitable for formation, stabilization and cleavage of a DNA—topoisomerase I covalent bond complex, such as physiologicalconditions, and the detection and measurement of enzyme activity, aswell as the preparation of cellular extracts are within the skill in theart (see also, the paragraph bridging pages 5-6, the fourth fullparagraph on page 11, and the Examples herein and Yang et al. (1996),supra).

EXAMPLES

The present invention is described further in the context of thefollowing examples. These examples serve to illustrate further thepresent invention and are not intended to limits its scope in any way.

In the following examples using yeast, standard protocols of yeastgrowth, mutagenesis, mating and sporulation were used (see, e.g.,Sherman, Methods Enzymol. 194:3 (1991); Treco and Lundblad, in CurrentProtocols in Molecular Biology, Ausubel et al., eds. (Wiley, N.Y.(1991)), vol. 2. pp. 13.1.1-13.1.7.).

Example 1

This example describes how the genomic DNA sequence of the yeast TDP1gene was obtained.

Extracts from colonies of chemically mutagenized Saccharomyces cervisiaewere assayed for TDP activity. A single strain, KYY337, had very low TDPactivity. In backcrosses to the parental line, the enzyme defectappeared to reflect a single mutation (designated enz). That is, when adiploid between the parental line and a defective line was sporulatedand haploid colonies were assayed at random, approximately equal numberswere found with normal and with low enzyme activity.

The strains were compared for sensitivity to killing by camptothecin.Despite the dramatic difference in TDP activity, the parental line andthe backcrossed enz mutant were insensitive to camptothecin. Whencombined with a disruption of the RAD9 gene, the camptothecinsensitivity of the low activity mutant (strain HNY244) was increased bya factor of 12 relative to the rad9 derivative of the parental strainHNY243. The same difference was observed after the mutant had undergonetwo additional rounds of backcrossing. In order to confirm thatcamptothecin-induced damage was due to topoisomerase trapping, the TOP1gene of HNY244 was disrupted and survival increased nearly 1,000-fold.

While the mutant line was sensitized to killing by camptothecin, themutant line was not sensitized to all sources of DNA damage. Forexample, the mutant line was not sensitized to killing by methyl methanesulfonate, a DNA-alkylating agent. In addition, independentoverexpression of two mutant yeast topoisomerase I genes that depressresealing of DNA, thereby leading to an accumulation of covalentcomplexes, were more toxic in a strain with low TDP activity than in acorresponding control strain.

In view of the above, a library of yeast genomic fragments was screenedfor the ability to improve the camptothecin resistance of HNY244 andrestore its TDP activity. The cloning scheme was based on the assessmentthat (i) the signal:noise ratio of the TDP assay would permit detectionof one positive colony in a group of 5-10 mutants and (ii) one cycle ofcamptothecin killing would enrich a positive colony by approximatelyten-fold.

Strain HNY244 was transformed by electroporation with a genomic librarythat had been made in a low-copy number vector (Rose et al., Gene 60:237 (1987)). Transformants were picked from uracil-selective plates andpooled in groups of about 50.

Each pool was separately grown and treated with camptothecin for 24hours. Cells were grown to near-saturation in medium with glycerol inplace of dextrose (YPG) to ensure a starting population with few or nopetite derivatives in accordance with standard methods. These cells wereresuspended at OD₆₅₀=0.4 in YPD (bacto-yeast extract, peptone anddextrose standard yeast growth medium), grown for 2 hrs and dilutedagain in YPD to OD₆₅₀=0.4. Drug was then added and samples werewithdrawn immediately and after 24 hrs at 30° C. After dilution andplating on YPD, surviving colonies were counted after 3-4 days ofgrowth. When plasmid-containing strains were assessed for camptothecinsensitivity, YPD was replaced throughout by uracil-deficient minimalmedium to ensure plasmid retention. The survivors were amplified bygrowth in YPD.

An extract of an aliquot of the resulting cells was assayed for TDPactivity. From 30 such pools, one was identified that had increasedactivity. Growth and assay of 15 colonies from this pool identified asingle clone. L10-13, with nearly wild-type levels of activity. DNAsequence from the insert of the plasmid in L10-13 placed itscentromere-distal end at coordinate 673926 of chromosome 11.

Several subclones of the approximately 8 kb insert in this plasmid weregenerated. Plasmid pNS2 was made from pL 10-13 by elimination of aNotI/SaII fragment. Elimination of an AatII-XbaI fragment from pNS2yielded pAXb, which has a 3.2 kb insert. Several subclones retained fullactivity.

Transformation of HNY244 with pNS2 or pAXb restored TDP activity andimproved camptothecin resistance. A control plasmid, pX1, that failed torestore TDP activity was made by removal of the central XbaI fragmentfrom pL10-13.

The smallest subclone contained a single open reading frame (ORF),namely YBR223c, which encodes a protein of 544 amino acids with amolecular weight of about 62,000.

A disruption that removed all but the first 32 amino acids of the ORFwas generated by PCR (Baudin, Nucleic Acids Res. 21: 3329 (1993);Ozier-Kalogeropoulos et al., Nucleic Acids Res. 21: 3329 (1993);Brachman et al., Yeast 14:115 (1998)) in strain HNY243. The resultingstrain had an enzymatic defect and camptothecin sensitivity was verysimilar to that of HNY244, indicating that YBR223c is involved in TDPactivity.

In order to distinguish whether YBR223c encodes or controls TDPactivity, a histidine-tagged version of YBR223c was introduced into E.coli, which, by itself, has no detectable TDP activity. A PCR fragmentcontaining the entire ORF YBR223c was cloned into the BamHI site ofpET15b. The resulting plasmid, pHN1856, was transformed into strainBL21(DE3) (Novagen, Madison, Wis.). Bacterial pellets from 3 liters of aculture that had been induced for 2 hrs were resuspended in 100 ml ofdisruption buffer (Yang et al., PNAS USA 93: 11534 (1996)), sonicated(7×3 min), clarified by centrifugation at 20,000 g, and assayed asdescribed above.

Induction of bacteria bearing this construct (but not a controlconstruct) resulted in massive overproduction of TDP, since crudeextracts of such cells had a specific activity greater than 10,000-foldhigher than that of extracts from a standard yeast strain. Moreover,most of the induced activity was bound to a tag-specific column.Specific elution released more than 75% of the bound activity, resultingin a fraction with a single Coomasie-stainable band of the expectedmolecular size. Based on the above, it was concluded that YBR223cencodes TDP1.

Database searches failed to reveal homology between TDP1 and any genesof known function. Even individualized comparisons to motifs identifiedin various phosphodiesterases and phosphatases were, at best, marginal.Thus, TDP1 encodes a novel enzyme. Eukaryotic databases contain severalunannotated sequences that match TDP1. The complete genome sequence ofthe nematode Caenorhabditis elegans contains a single ORF withsignificant similarity to TDP1. Probing EST databases with the yeast andnematode proteins revealed many significant matches (see FIG. 5, whichis an alignment of TDP homologs from various organisms, in which “hs” isthe human deduced amino acid sequence (SEQ ID NO: 5), “mm” is the mouseamino acid sequence (Mus muscularis; assembly of mouse ESTs GenBankAA940134, W89267 and W13117) (SEQ ID NO: 6), “dm” is the fruit flydeduced amino acid sequence (Drosophila melanogaster; GenBank Al517253)(SEQ ID NO: 7), “ce” is the nematode deduced amino acid sequence(Caenorhabditis elegans (ce; gene F52C12.1; GenBank AF100657.2)) (SEQ IDNO: 8), “sp” is the Schitosaccharomyces pombe (Sanger Centre SequencingGroup (Cambridge, UK) deduced amino acid sequence (SEQ ID NO: 9), and“sc” is the yeast deduced amino acid sequence (Saccharomyces cerevisiae;gene YBR223c; GenBank Z36092.1) (SEQ ID NO: 4). Black boxes indicateidentities, whereas shaded boxes indicate similarities and “x” indicatesuncertainty in the GenBank entry AA48921. X's were confirmed by sequenceanalysis of the product of a 3′ RACE of a human EST that showed that thesequence in the region of ambiguity is identical to that shown for themouse).

Example 2

This example describes how the cDNA sequence of the human TDP1 gene wasobtained.

A human database and a mouse EST database were searched with the yeastsequence and several EST's were identified that could be aligned to makeup a single ORF with substantial similarity to the carboxy-terminal halfof TDP1. In order to determine if the homology extends further, PCR wasperformed on a collection of human cDNAs (Marathon-Ready; ClontechLaboratories, Palo Alto, Calif.) with a primer complementary to an ESTsequence identified in the human EST database and a primer complementaryto the tag affixed to the 5′ end of the cDNAs. The resulting 5′-RACEproducts were cloned. The sequence of one of the longest clones alignedwell to most of the 5′ half of the yeast and nematode ORFs. Based on theabove, it was concluded that the TDP1 gene is highly conserved ineukaryotes.

Partial 5′ and 3′ sequences of the human TDP1 have been deposited asGenBank AF182002 and AF182003, respectively. The complete cDNA sequence(SEQ ID NO: 1) is shown in FIG. 1. The deduced amino acid sequence (SEQID NO: 13) is shown in FIG. 2.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be apparent to those of ordinary skill in the artthat variations in the preferred embodiments can be prepared and usedand that the invention can be practiced otherwise than as specificallydescribed herein. The present invention is intended to include suchvariations and alternative practices. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the following claims.

1. An isolated or purified nucleic acid molecule comprising SEQ IDNO:
 1. 2. A vector comprising the nucleic acid molecule of claim
 1. 3.An isolated host cell comprising the vector of claim
 2. 4. An isolatedor purified nucleic acid molecule comprising a nucleic acid sequencethat is at least 95% identical to SEQ ID NO: 1 that encodes apolypeptide having tyrosine-DNA phosphodiesterase activity.
 5. Anisolated or purified nucleic acid molecule encoding the amino acidsequence of SEQ ID NO:
 2. 6. An isolated or purified nucleic acidmolecule having at least 95% sequence identity to the nucleic acidmolecule of claim 5 and encoding a polypeptide having tyrosine-DNAphosphodiesterase activity.
 7. An isolated or purified RNA moleculecomprising a nucleic acid sequence transcribed from a nucleic acidsequence encoding the amino acid sequence of SEQ ID NO:
 2. 8. Theisolated or purified RNA molecule of claim 7, wherein the nucleic acidsequence is transcribed from SEQ ID NO:
 1. 9. A vector comprising theRNA molecule of claim
 7. 10. An isolated host cell comprising the vectorof claim
 9. 11. An isolated or purified nucleic acid molecule consistingof a continuous fragment of SEQ ID NO: 1, wherein said fragment encodesa polypeptide having tyrosine-DNA phosphodiesterase activity.
 12. Avector comprising the nucleic acid molecule of claim
 11. 13. An isolatedhost cell comprising the vector of claim
 12. 14. The isolated orpurified nucleic acid molecule of claim 11, wherein the nucleic acidmolecule encodes the amino acid sequence of SEQ ID NO: 2.